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A kind of digestion solution and separation method of primary separation of tendon stem cells derived from Achilles tendon

A technology of tendon stem cells and separation method, which is applied in the field of primary separation and separation of Achilles tendon-derived tendon stem cells, can solve the problems of long digestion time, low separation rate, and lack of cell state, and achieves high digestion efficiency and reduced Physical damage, the effect of shortening digestion time

Active Publication Date: 2019-07-19
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, if the grinding force of the mechanical grinding method is too large, the target cells will be seriously damaged, while the force is too small, it will take a long time to grind, and finally the ideal cell state will not be obtained, and the separation rate will be low.
The digestion conditions of the enzymatic digestion method are relatively mild, but the digestion time is very long

Method used

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  • A kind of digestion solution and separation method of primary separation of tendon stem cells derived from Achilles tendon
  • A kind of digestion solution and separation method of primary separation of tendon stem cells derived from Achilles tendon
  • A kind of digestion solution and separation method of primary separation of tendon stem cells derived from Achilles tendon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Human Achilles Tendon-derived Tendon Stem Cell Primary Separation Digestive Solution and Separation Method

[0032] Pretreatment of human Achilles tendon tissue: wash human Achilles tendon tissue with PBS for 3 times in a sterile environment, cut 5g of human Achilles tendon tissue into tissue fragments with a size of about 1mm×1mm×1mm; human Achilles tendon tissue comes from a patient with tendon rupture Achilles tendon tissue trimmed during Achilles tendon suture repair;

[0033] S1. Mix 500 mg of human Achilles tendon tissue fragments with 6 mL of digestive juice, and shake and digest at 37° C. for 30 minutes; the digestive juice is a type I collagenase solution containing a surfactant, and the concentration of type I collagenase in the digestive juice is 3mg / mL, the concentration of surfactant and the shaking speed are set as shown in Table 1;

[0034] S2. Centrifuge at 1500rpm for 10min to stop the digestion, discard the supernatant, resuspend the pellet...

Embodiment 2

[0043] Example 2. Morphological observation of tendon stem cells derived from human Achilles tendon

[0044] Microscopically observe the cell morphology in the process of isolating human Achilles tendon-derived tendon stem cells by the primary isolation method of Achilles tendon-derived tendon stem cells of the present invention, in the primary culture process, and in the subculture process of the isolated human Achilles tendon-derived tendon stem cells . Nucleated cells obtained from human Achilles tendon tissue digestion, the cells grow in a clonal manner. Multiple scattered clustered cell colonies can be seen in the primary culture, growing on the wall in a radial-like manner from the center ( figure 2 ), the cell shape is irregular, mainly polygonal, the nucleus gathers in the center, and the protrusions formed by the cytoplasm radially outward ( image 3 , 40 times image), conforming to the morphological characteristics of tendon stem cells derived from human Achilles ...

Embodiment 3

[0045] Example 3, flow cytometry detection of cellular immune phenotype

[0046] The human Achilles tendon-derived tendon stem cells isolated from group G11 in Example 1 were subcultured in a 37°C, 5% carbon dioxide incubator, and the subculture medium was L-DMEM medium containing 10% FBS by volume. Take the P3 generation cells for cell immunophenotype detection, the specific method is: collect about 2×10 6 P3 generation cells were centrifuged at 1500rpm for 4min, discarded the supernatant, resuspended in Buffer solution, pipetted 100μL of the cell suspension into the Ep tube, added anti-CD90, CD44, CD106, CD34 and incubated for 30min in the dark. After centrifugation at 1500rpm for 4min, the supernatant was discarded. Add 300 μL Buffer solution to each sample tube to resuspend, and test on the machine.

[0047] The results of cell surface antigen identification by flow cytometry further showed that tendon stem cells derived from human Achilles tendon had clear stem cell cha...

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Abstract

The invention discloses digestive juice for primary-generation separation of heel tendon source muscle tendon stem cells and a separation method. The digestive juice for separation is an enzyme solution containing a surfactant, and the digestive juice is an I-type collagenase solution containing a surfactant. The separation method includes the step that the heel tendon tissue undergoes oscillation digestion by using the digestive juice, wherein the digestive juice is the enzyme solution containing the surfactant. When the digestive juice is used for digesting larger-hardness tissue blocks, and a shear force suffered by the cells in the oscillation digestion process is reduced and a good cell state is ensured by using the digestive juice containing the surfactant. The digestion time can be effectively shortened through oscillation digestion, small heel tendon tissue blocks can be obtained only by needing 30 minutes, and the digestion efficiency is high.

Description

technical field [0001] The present invention relates to the field of isolation and culture of stem cells, in particular to a primary isolation digestive solution and isolation method of tendon stem cells derived from Achilles tendon. Background technique [0002] Tendons are strong, fibrous connective tissues that connect bones to muscles. Tendinitis usually refers to the degeneration of tendon collagen fibers caused by excessive use of muscle fibers and repeated strong stretching. In addition to involving the tendon itself, it can also involve the tendon sheath. In the past, tendinitis was commonly referred to as tendonitis, but in fact tendonitis is not a single inflammation. In most cases, it is often combined with degeneration of the collagen tissue of the affected tendon, so it is now commonly called tendinopathy. [0003] Tendinopathy is common in people who exercise heavily, and is characterized clinically by localized pain, swelling, and dysfunction. The common dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
Inventor 陈海佳王一飞葛啸虎黄幸李平
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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